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J Bacteriol. 2010 August; 192(15): 4074–4075.
Published online 2010 June 4. doi:  10.1128/JB.00562-10
PMCID: PMC2916366

Complete Genome Sequence of Bacillus thuringiensis Mutant Strain BMB171 [down-pointing small open triangle]


Bacillus thuringiensis has been widely used as a biopesticide for a long time. Here we report the finished and annotated genome sequence of B. thuringiensis mutant strain BMB171, an acrystalliferous mutant strain with a high transformation frequency obtained and stocked in our laboratory.

Bacillus thuringiensis is an insect pathogen which is widely used as a biopesticide due to its various endogenous crystal proteins and spores (12). To improve the virulence and practical effectiveness of B. thuringiensis, genetic transformation of different genes with beneficial traits is a fundamental procedure. Simultaneously, genetic transformation can facilitate functional genomic research. However, wild-type strains are not suitable to be used as recipient strains because of low transformation efficiency. This obstacle is mainly caused by the thick cell wall layer of B. thuringiensis together with multiple plasmids inside the cell, which harbor genes encoding insecticidal crystal proteins. We used the method of elevating the growth temperature and adding 0.05% sodium dodecyl sulfate to treat several parental strains and finally obtained mutant strain BMB171, with no resident plasmid, from wild-type crystalliferous strain YBT-1463 (9). The electrotransformation frequency of mutant BMB171 could reach up to 107 transformants/μg DNA after optimization of the electrotransformation parameters (7), which was 4.8 × 104-fold higher than that of the parental strain (8). Moreover, mutant strain BMB171 exhibited the same characteristics as YBT-1463, such as metabolic abilities and growth properties, as well as sensitivity to 10 antibiotics (8). Of course, BMB171 could produce parasporal crystals with characteristic geometric shapes through the expression of relevant cry genes carried by plasmids (7). Thus, B. thuringiensis mutant strain BMB171 has become a major recipient strain and is widely used for insecticidal crystal protein-encoding gene expression (14, 15), cell surface display (10, 13), gene function and regulation researches (2, 5), etc.

The B. thuringiensis mutant strain BMB171 genome was sequenced by using a massive parallel pyrosequencing technology (454 GS-FLX). A total of 448,963 high-quality reads with an average read length of 391 bp were produced, providing about 32-fold coverage of the genome. Assembly was performed using the Newbler software of the 454 suite package (454 Life Sciences), which resulted in 193 large (defined as >500 bp) contigs. The relationship of contigs was determined by multiplex PCR, and gaps were filled through sequencing of PCR products by primer walking or shotgun sequencing with an ABI 3730 sequencer. The Phred/Phrap/Consed software package (3) was used for final sequence assembly and quality assessment. Protein-coding genes were predicted by combining the results of Glimmer 3.02 (1) and ZCURVE (4), followed by manual inspection. Both tRNA and rRNA genes were identified by tRNAscan-SE (11) and RNAmmer (6), respectively. Functional annotation was performed by searching against a protein database of the microbial genome developed in house.

The 5.64-Mb genome of B. thuringiensis mutant strain BMB171 contains two replicons: a circular chromosome (5.33 Mb) encoding 5,088 open reading frames (ORFs) and a circular plasmid (0.31 Mb), which is named pBMB171, encoding 276 predicted ORFs. The G+C content of the chromosome is 35.3%, while that of the plasmid is 33.3%. The mutant strain BMB171 genome encodes 104 tRNAs and 14 rRNA operons. A previous study indicated that BMB171 is a plasmid-free mutant (9); however, our sequencing results demonstrated that a large plasmid still remains. The reason why the plasmid was not detected previously might be its large size and low copy number. We did not find any crystal protein genes in either chromosome or plasmid sequences, which was consistent with previous observations (9).

In summary, the complete B. thuringiensis mutant strain BMB171 genome provides a better-defined genetic background for gene expression and regulation studies, especially crystal protein production and metabolic network construction.

Nucleotide sequence accession numbers.

The sequence and annotation data for B. thuringiensis mutant strain BMB171 have been deposited in GenBank under accession numbers CP001903 (chromosome) and CP001904 (plasmid).


This research was supported by the Chinese National Natural Science Funds (grants 30930004, 30770022, and 30900016) and by the National High Technology Research and Development Program of China (863 Program, grant 2008AA02Z112).


[down-pointing small open triangle]Published ahead of print on 4 June 2010.


1. Delcher, A. L., K. A. Bratke, E. C. Powers, and S. L. Salzberg. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673-679. [PMC free article] [PubMed]
2. Fang, S., L. Wang, W. Guo, X. Zhang, D. Peng, C. Luo, Z. Yu, and M. Sun. 2009. Bacillus thuringiensis Bel protein enhances the toxicity of Cry1Ac protein to Helicoverpa armigera larvae by degrading insect intestinal mucin. Appl. Environ. Microbiol. 75:5237-5243. [PMC free article] [PubMed]
3. Gordon, D., C. Desmarais, and P. Green. 2001. Automated finishing with Autofinish. Genome Res. 11:614-625. [PubMed]
4. Guo, F., H. Ou, and C. Zhang. 2003. ZCURVE: a new system for recognizing protein-coding genes in bacterial and archaeal genomes. Nucleic Acids Res. 31:1780-1789. [PMC free article] [PubMed]
5. Ji, F., Y. Zhu, S. Ju, R. Zhang, Z. Yu, and M. Sun. 2009. Promoters of crystal protein genes do not control crystal formation inside exposporium of Bacillus thuringiensis ssp. finitimus strain YBT-1520. FEMS Microbiol. Lett. 300:11-17. [PubMed]
6. Lagesen, K., P. F. Hallin, E. A. Rodland, H. H. Staerfeldt, T. Rognes, and D. W. Ussery. 2007. RNAmmer: consistent annotation of rRNA genes in genomic sequences. Nucleic Acids Res. 35:3100-3108. [PMC free article] [PubMed]
7. Li, L., Z. Shao, and Z. Yu. 2000. Transformation of Bacillus thuringiensis recipient BMB171 by electroporation. Wei Sheng Wu Xue Tong Bao 27:331-334. (In Chinese.)
8. Li, L., Z. Wang, and Z. Yu. 2000. Influence on properties of Bacillus thuringiensis YBT-1463 strain by curing its resident plasmids. Wei Sheng Wu Xue Tong Bao 27:25-28. (In Chinese.)
9. Li, L., C. Yang, Z. Liu, F. Li, and Z. Yu. 2000. Screening of acrystalliferous mutants from Bacillus thuringiensis and their transformation properties. Wei Sheng Wu Xue Bao 40:85-90. (In Chinese.) [PubMed]
10. Liu, M., S. Li, S. Hu, C. Zhao, D. Bi, and M. Sun. 2008. Display of avian influenza virus nucleoprotein on Bacillus thuringiensis cell surface using CTC as a fusion partner. Appl. Microbiol. Biotechnol. 78:669-676. [PubMed]
11. Lowe, T. M., and S. R. Eddy. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25:955-964. [PMC free article] [PubMed]
12. Schnepf, E., N. Crichmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D. R. Zeigler, and D. H. Dean. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:775-806. [PMC free article] [PubMed]
13. Shao, X., M. Jiang, Z. Yu, H. Cai, and L. Li. 2009. Surface display of heterologous proteins in Bacillus thuringiensis using a peptidoglycan hydrolase anchor. Microb. Cell Fact. 8:48. doi:.10.1186/1475-2859-8-48 [PMC free article] [PubMed] [Cross Ref]
14. Zhu, C., L. Ruan, D. Peng, Z. Yu, and M. Sun. 2006. Vegetative insecticidal protein enhancing the toxicity of Bacillus thuringiensis kurstaki against Spodoptera exigua. Lett. Appl. Microbiol. 42:109-114. [PubMed]
15. Zhu, C., and Z. Yu. 2008. The surface layer protein of Bacillus thuringiensis CTC forms unique intracellular parasporal inclusion body. J. Basic Microbiol. 48:302-307. [PubMed]

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